PROBING OF THE LOCATION OF THE ALLOSTERIC SITE ON M1 MUSCARINIC RECEPTORS BY SITE-DIRECTED MUTAGENESIS

In an attempt to locate the allosteric site on muscarinic receptors to which gallamine binds, 21 residues in the putative external loops and loop/transmembrane helix interfaces have been mutated to alanine. The se residues are conserved in mammalian m1-m5 receptors. All mutant rec eptors can be expressed in COS-7 cells at high levels and appear to be functional, in that acetylcholine binding is sensitive to GTP. The ga llamine binding site does not appear to involve the first, second, and most of the third extracellular loops. Tryptophan-400 and -101 inhibi t gallamine binding when mutated to alanine or to phenylalanine and ma y form part of the allosteric site. Several mutations also affect anta gonist binding. Surprisingly, tryptophan-91, a residue conserved in mo noamine and peptide receptors, is important for antagonist binding. Th is residue, present in the middle of the first extracellular loop, may have a structural role in many G protein-coupled receptors. Antagonis t binding is also affected by mutations of tryptophan-101 and tyrosine -404 to alanine or phenylalanine. In a helical wheel model, tryptophan -101 and tyrosine-404, in conjunction with serine-78, aspartate-105, a nd tyrosine-408, form a cluster of residues that have been reported to affect antagonist binding when mutated, and they may therefore be par t of the antagonist binding site. It is suggested that the allosteric site may be located close to and just extracellular to the antagonist binding site. The binding of methoctramine, an antagonist with alloste ric properties, is not substantially affected by mutations at tryptoph an-91, -101, and -400 and tyrosine-404, and thus these amino acids are not important for its binding. The binding of himbacine, another anta gonist with allosteric properties, is affected by these mutations but in a manner different from that of gallamine or competitive antagonist s. It has not been possible to determine whether methoctramine and him bacine bind exclusively to the allosteric site or to both the competit ive site and the allosteric site.